Detecting Enzyme Activity A Case Study of Polygalacturonase

The two methods described herein are inherently different in that one is a traditional initial velocity assay that attempts to quantitatively measure rates of product formation (see Basic Protocol 1), whereas the other correlates the activity of an enzyme preparation with its ability to change the rheological properties (i.e., viscosity) of a substrate solution (see Basic Protocol 2). For both assays, it is presumed that the analyst is using soluble substrate and enzyme preparations, appropriate buffer systems, and a method to control the reaction mixture temperature. The ultimate goal of both assays is the same to obtain a quantitative estimate of the PGase activity of a test solution. 

The substrate prescribed for these assays is commercially available polygalacturonic acid. It was chosen based on its availability and on the observation that PGase enzymes are typically specific for glycosidic linkages between de-esterified galacturonic acid units 

The reaction conditions chosen for the assays are based on published optimal conditions for PGase enzymes. These enzymes typically have maximal activities at slightly acidic pH (Tucker and Seymour, 2002) and, in general, appear to be relatively stable at temperatures from 30° to 40°C. Optimal reaction conditions are likely to be enzyme specific, so one may have to alter the conditions to match the properties of the enzyme of interest. In all cases, the analyst should take into account the properties of the substrate, particularly its solubility, as well as the properties of the enzyme. For example, because solutions of polygalacturonic acid tend to gel as the pH is lowered below 3, viscometric assays (Basic Protocol 2) at these relatively low pHs are often not feasible. 

The PGase extraction scheme outlined below (see Support Protocol) is based on the properties of the enzyme from tomato (Pressey, 1986). Enzymes from other sources will probably require different extraction conditions. A more detailed discussion of common approaches to enzyme extraction is included (see Background Information, discussion of samples for pectic enzyme assays). 

Detecting Enzyme Activity A Case Study of Polygalacturonase 

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Cl.] Expression and Measurement of Enzyme Activity C1.2 Detecting Enzyme Activity A Case Study of Polygalacturonase... 

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